*Target (protein/gene name): Serine/Threonine- Protein Kinase B PkNB *RefSeq#: NC 015848.1 , WP 003400356.1 *Protein ID (NP or XP #) or Wolbachia#: G0TLP7-1 *Organism (including strain): Mycobacterium canettii (strain CIPT 140010059) (complex of Mycobacterium tuberculosis) Etiologic Risk Group (see link below): RG2
Disease Information Mycobacterium tuberculosis is an obligate pathogenic bacterial species that causes tuberculosis. Humans are the only known reservoirs of M. tuberculosis and can be spread through air droplets originating from a person who has the disease either coughing, sneezing, speaking, or singing. Tuberculosis is one of the oldest human diseases and remains a major cause of death in some countries. Tuberculosis can affect bone, central nervous system, and other vital organ systems but is a pulmonary disease caused by Mycobacterium tuberculosis transmitted through the air. New strains of Mycobacterium tuberculosis appear often that are more virulent and require new vaccines and drugs. Mycobacterium Canetti is very similar to Mycobacterium tuberculosis and belongs to both belong to the Mycobacterium tuberculosis complex(MTBC) which is a group of genetically similar Mycobacterium species that can cause tuberculosis in living things.
Essentiality of this protein: The Ser/Thr protein Kinase (STPK) is essential for the Mycobacterium Canettii to survive because it plays an important role in regulating growth and division. STPK mediates protein phosphorylation which is essential for cells to transduce extracellular signals into responses. Ser/Thr kinase activity has numerous effects on the cells that can affect transcription of DNA and immune defense. Without the precise functioning of the kinase the accurate signal to control the growth of cells will not be transduced in the signal pathway.
Figure 1. Reaction Mechanism produced by the ser/thr protein Kinase from BRENDA. Enzyme Assay information :
Array-Based Fluorescence Assay -- to paper that contains assay information: http://www.brenda-enzymes.org/literature.php?e=2.7.11.1&r=691233 -- links to assay reagents (substrates) pages. --- Lists cost and quantity of substrate reagents, supplier, and catalog #
Structure (PDB or Homology model) -- PDB # : 1MRU Similarity to Humans: After running BLASTP with the amino acid sequence of STPK there was a match of 81% to human STPKs. Current Inhibitors: Inhibiting the inducible dimerization system has been looked into in this paper.
Expression Information : The protein can and has been expressed in E.Coli bacteria. Paper Describing expression of the protein. Purification Method: Purified by washing in a solution of 10% B-Per reagent and centrifuging. Protein can be separated with sodium dodecyl sulfate. Purification Process.
Image of protein (PyMol with features delineated and shown separately): Figure 2. Protein structure of 1MUR from PDB. *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
Chain A Sequence:
GSHMTTPSHLSDRYELGEILGFGGMSEVHLARDLRLHRDVAVKVLRADLARDPSFYLRFRREAQNAAALNHPAIVAVYDT
*length of your protein in Amino Acids: 644 Molecular Weight: 69555.03 Extinction coefficient: 30175 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Figure 3. TMpred Graph of the STPK protein. CDS Gene Sequence: ATGAGCGACGTGGCTATTGTGAAGGAGGGTTGGCTGCACAAACGAGGGGAGTACATCAAGACCTGGCGGC CACGCTACTTCCTCCTCAAGAATGATGGCACCTTCATTGGCTACAAGGAGCGGCCGCAGGATGTGGACCA ACGTGAGGCTCCCCTCAACAACTTCTCTGTGGCGCAGTGCCAGCTGATGAAGACGGAGCGGCCCCGGCCC AACACCTTCATCATCCGCTGCCTGCAGTGGACCACTGTCATCGAACGCACCTTCCATGTGGAGACTCCTG AGGAGCGGGAGGAGTGGACAACCGCCATCCAGACTGTGGCTGACGGCCTCAAGAAGCAGGAGGAGGAGGA GATGGACTTCCGGTCGGGCTCACCCAGTGACAACTCAGGGGCTGAAGAGATGGAGGTGTCCCTGGCCAAG CCCAAGCACCGCGTGACCATGAACGAGTTTGAGTACCTGAAGCTGCTGGGCAAGGGCACTTTCGGCAAGG TGATCCTGGTGAAGGAGAAGGCCACAGGCCGCTACTACGCCATGAAGATCCTCAAGAAGGAAGTCATCGT GGCCAAGGACGAGGTGGCCCACACACTCACCGAGAACCGCGTCCTGCAGAACTCCAGGCACCCCTTCCTC ACAGCCCTGAAGTACTCTTTCCAGACCCACGACCGCCTCTGCTTTGTCATGGAGTACGCCAACGGGGGCG AGCTGTTCTTCCACCTGTCCCGGGAGCGTGTGTTCTCCGAGGACCGGGCCCGCTTCTATGGCGCTGAGAT TGTGTCAGCCCTGGACTACCTGCACTCGGAGAAGAACGTGGTGTACCGGGACCTCAAGCTGGAGAACCTC ATGCTGGACAAGGACGGGCACATTAAGATCACAGACTTCGGGCTGTGCAAGGAGGGGATCAAGGACGGTG CCACCATGAAGACCTTTTGCGGCACACCTGAGTACCTGGCCCCCGAGGTGCTGGAGGACAATGACTACGG CCGTGCAGTGGACTGGTGGGGGCTGGGCGTGGTCATGTACGAGATGATGTGCGGTCGCCTGCCCTTCTAC AACCAGGACCATGAGAAGCTTTTTGAGCTCATCCTCATGGAGGAGATCCGCTTCCCGCGCACGCTTGGTC CCGAGGCCAAGTCCTTGCTTTCAGGGCTGCTCAAGAAGGACCCCAAGCAGAGGCTTGGCGGGGGCTCCGA GGACGCCAAGGAGATCATGCAGCATCGCTTCTTTGCCGGTATCGTGTGGCAGCACGTGTACGAGAAGAAG CTCAGCCCACCCTTCAAGCCCCAGGTCACGTCGGAGACTGACACCAGGTATTTTGATGAGGAGTTCACGG CCCAGATGATCACCATCACACCACCTGACCAAGATGACAGCATGGAGTGTGTGGACAGCGAGCGCAGGCC CCACTTCCCCCAGTTCTCCTACTCGGCCAGCGGCACGGCCTGA
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences): **
*RefSeq#: NC 015848.1 , WP 003400356.1
*Protein ID (NP or XP #) or Wolbachia#: G0TLP7-1
*Organism (including strain): Mycobacterium canettii (strain CIPT 140010059) (complex of Mycobacterium tuberculosis)
Etiologic Risk Group (see link below): RG2
Disease Information
Mycobacterium tuberculosis is an obligate pathogenic bacterial species that causes tuberculosis. Humans are the only known reservoirs of M. tuberculosis and can be spread through air droplets originating from a person who has the disease either coughing, sneezing, speaking, or singing. Tuberculosis is one of the oldest human diseases and remains a major cause of death in some countries. Tuberculosis can affect bone, central nervous system, and other vital organ systems but is a pulmonary disease caused by Mycobacterium tuberculosis transmitted through the air. New strains of Mycobacterium tuberculosis appear often that are more virulent and require new vaccines and drugs. Mycobacterium Canetti is very similar to Mycobacterium tuberculosis and belongs to both belong to the Mycobacterium tuberculosis complex(MTBC) which is a group of genetically similar Mycobacterium species that can cause tuberculosis in living things.
Link to TDR Targets page (if present): http://www.tdrtargets.org/targets/view?gene_id=6795
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): UniProt: http://www.uniprot.org/uniprot/G0TLP7
Essentiality of this protein:
The Ser/Thr protein Kinase (STPK) is essential for the Mycobacterium Canettii to survive because it plays an important role in regulating growth and division. STPK mediates protein phosphorylation which is essential for cells to transduce extracellular signals into responses. Ser/Thr kinase activity has numerous effects on the cells that can affect transcription of DNA and immune defense. Without the precise functioning of the kinase the accurate signal to control the growth of cells will not be transduced in the signal pathway.
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): The structure appears to be monomeric
Complex of proteins: Functions with a Protein Kinase Domain and works with protein kinases
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
On Druggability Index (range 0-1): 0.7
The structure of PknB in complex with mitoxantrone, an ATP-competitive inhibitor, suggests a mode of protein kinase regulation in mycobacteria
*EC#: 2.7.11.1
Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=2.7.11.1
Figure 1. Reaction Mechanism produced by the ser/thr protein Kinase from BRENDA.
Enzyme Assay information :
Array-Based Fluorescence Assay
-- to paper that contains assay information: http://www.brenda-enzymes.org/literature.php?e=2.7.11.1&r=691233
-- links to assay reagents (substrates) pages.
--- Lists cost and quantity of substrate reagents, supplier, and catalog #
Structure (PDB or Homology model)
-- PDB # : 1MRU
Similarity to Humans:
After running BLASTP with the amino acid sequence of STPK there was a match of 81% to human STPKs.
Current Inhibitors:
Inhibiting the inducible dimerization system has been looked into in this paper.
Expression Information : The protein can and has been expressed in E.Coli bacteria. Paper Describing expression of the protein.
Purification Method: Purified by washing in a solution of 10% B-Per reagent and centrifuging. Protein can be separated with sodium dodecyl sulfate. Purification Process.
Image of protein (PyMol with features delineated and shown separately):
Figure 2. Protein structure of 1MUR from PDB.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
Chain A Sequence:
GSHMTTPSHLSDRYELGEILGFGGMSEVHLARDLRLHRDVAVKVLRADLARDPSFYLRFRREAQNAAALNHPAIVAVYDT
GEAETPAGPLPYIVMEYVDGVTLRDIVHTEGPMTPKRAIEVIADACQALNFSHQNGIIHRDVKPANIMISATNAVKVMDF
GIARAIADSGNSVTQTAAVIGTAQYLSPEQARGDSVDARSDVYSLGCVLYEVLTGEPPFTGDSPVSVAYQHVREDPIPPS
ARHEGLSADLDAVVLKALAKNPENRYQTAAEMRADLVRVHNGEPPEAPKVLTDAERTSLLSSAAGNLSGPR
Chain B Sequence:
GSHMTTPSHLSDRYELGEILGFGGMSEVHLARDLRLHRDVAVKVLRADLARDPSFYLRFRREAQNAAALNHPAIVAVYDT
GEAETPAGPLPYIVMEYVDGVTLRDIVHTEGPMTPKRAIEVIADACQALNFSHQNGIIHRDVKPANIMISATNAVKVMDF
GIARAIADSGNSVTQTAAVIGTAQYLSPEQARGDSVDARSDVYSLGCVLYEVLTGEPPFTGDSPVSVAYQHVREDPIPPS
ARHEGLSADLDAVVLKALAKNPENRYQTAAEMRADLVRVHNGEPPEAPKVLTDAERTSLLSSAAGNLSGPR
*length of your protein in Amino Acids: 644
Molecular Weight: 69555.03
Extinction coefficient: 30175
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html).
Figure 3. TMpred Graph of the STPK protein.
CDS Gene Sequence:
ATGAGCGACGTGGCTATTGTGAAGGAGGGTTGGCTGCACAAACGAGGGGAGTACATCAAGACCTGGCGGC
CACGCTACTTCCTCCTCAAGAATGATGGCACCTTCATTGGCTACAAGGAGCGGCCGCAGGATGTGGACCA
ACGTGAGGCTCCCCTCAACAACTTCTCTGTGGCGCAGTGCCAGCTGATGAAGACGGAGCGGCCCCGGCCC
AACACCTTCATCATCCGCTGCCTGCAGTGGACCACTGTCATCGAACGCACCTTCCATGTGGAGACTCCTG
AGGAGCGGGAGGAGTGGACAACCGCCATCCAGACTGTGGCTGACGGCCTCAAGAAGCAGGAGGAGGAGGA
GATGGACTTCCGGTCGGGCTCACCCAGTGACAACTCAGGGGCTGAAGAGATGGAGGTGTCCCTGGCCAAG
CCCAAGCACCGCGTGACCATGAACGAGTTTGAGTACCTGAAGCTGCTGGGCAAGGGCACTTTCGGCAAGG
TGATCCTGGTGAAGGAGAAGGCCACAGGCCGCTACTACGCCATGAAGATCCTCAAGAAGGAAGTCATCGT
GGCCAAGGACGAGGTGGCCCACACACTCACCGAGAACCGCGTCCTGCAGAACTCCAGGCACCCCTTCCTC
ACAGCCCTGAAGTACTCTTTCCAGACCCACGACCGCCTCTGCTTTGTCATGGAGTACGCCAACGGGGGCG
AGCTGTTCTTCCACCTGTCCCGGGAGCGTGTGTTCTCCGAGGACCGGGCCCGCTTCTATGGCGCTGAGAT
TGTGTCAGCCCTGGACTACCTGCACTCGGAGAAGAACGTGGTGTACCGGGACCTCAAGCTGGAGAACCTC
ATGCTGGACAAGGACGGGCACATTAAGATCACAGACTTCGGGCTGTGCAAGGAGGGGATCAAGGACGGTG
CCACCATGAAGACCTTTTGCGGCACACCTGAGTACCTGGCCCCCGAGGTGCTGGAGGACAATGACTACGG
CCGTGCAGTGGACTGGTGGGGGCTGGGCGTGGTCATGTACGAGATGATGTGCGGTCGCCTGCCCTTCTAC
AACCAGGACCATGAGAAGCTTTTTGAGCTCATCCTCATGGAGGAGATCCGCTTCCCGCGCACGCTTGGTC
CCGAGGCCAAGTCCTTGCTTTCAGGGCTGCTCAAGAAGGACCCCAAGCAGAGGCTTGGCGGGGGCTCCGA
GGACGCCAAGGAGATCATGCAGCATCGCTTCTTTGCCGGTATCGTGTGGCAGCACGTGTACGAGAAGAAG
CTCAGCCCACCCTTCAAGCCCCAGGTCACGTCGGAGACTGACACCAGGTATTTTGATGAGGAGTTCACGG
CCCAGATGATCACCATCACACCACCTGACCAAGATGACAGCATGGAGTGTGTGGACAGCGAGCGCAGGCC
CCACTTCCCCCAGTTCTCCTACTCGGCCAGCGGCACGGCCTGA
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
**